PRESENTION BY;Okinyi J. AdamI56/10103/08(Kenyatta University)

SUPERVISORS;Dr. Runo S. MainaDepartment of Biochemistry and BiotechnologyKenyatta University

Dr. Charles A. O. MidegaDepartment of Plant HealthInternational Center of Insect Physiology and Ecology.

Molecular determination and characterization of phytoplasma 16S rRNA gene in selected wild

grasses from western Kenya

HypothesisBackground information

• Napier grass an indigenous tropical African clumping grass, is the major livestock feed in zero-grazing systems in western Kenya.

• In East Africa, Napier stunt phytoplasma poses a serious threat to Napier grass farming.

• The disease symptoms include severe stunted growth and loss of biomass

• It is mainly transmitted by a leafhopper Maeistas (=Recilia) banda in Kenya (Obura et al., 2009)

Photographs illustrating the comparison between Health (a) & Diseased (b) Napier grass.

a

b

Life cycle of NSD in the region

Recilia banda

Statement of the problem• Napier stunt disease has reduced Napier productivity by 30-90% in

the region • Phytoplasma attacks other wild grasses, it is likely that several wild

grasses could be infected by specific phytoplasma strains • These wild grasses might also act as reservoirs for fresh inoculums• The determination and characterization of phytoplasma 16sr RNA

gene in wild grasses from western Kenya is necessary for precise and sustainable phytoplasma disease management

BGWL HWLD NSD

Hypothesis

There is no diversity of phytoplasmas and wild grasses hosting phytoplasmas in western Kenya.

Objectives

General Objective• To characterize phytoplasmas, and describe their

host range among wild grasses in western Kenya.

Specific Objectives• To detect and identify phytoplasma strains infecting

wild grasses in western Kenya• To identify wild grass species hosting phytoplasmas

in western Kenya.

Study Area KEY:

Positive

Negative

Sampling strategy

QuadratTransects1m

1m

Grass field boundary

1-3m

1-3 m

Phytoplasma detection & Characterization

Collection of leaf samples (300mg)

DNA Extraction (CTAB)

PCR amplification P1/P6 then NapF/NapR

Purification of PCR products

genotypic Sequencing of PCR products

Sequence editing via BioEdit softwareComparison of

sequences using BLAST search at NCBI

Phylogenetic analysis by neighbour joining method (Saitou & Nei, 1987)

Symptomatic C. dactylon as observed in the field: (A) and (C) Chlorosis of above ground plant parts. (B) and (D) shortened

internodes, stunted growth and bushy plants.

A

C

B

D

Gel electrophoresis results of 1.0L purified PCR products obtained for direct sequencing

20001500

1000

500700

100

300

Unrooted bootstrap consensus tree illustrating the phylogenetic relationships for the 16Sr RNA genes of the phytoplasma strains derived from wild grasses in western Kenya.

Unrooted computed linearized phylogenetic tree assembled by the Neighbour-Joining method highlighting two major groups of phylogenetic relationships for phytoplasma isolates

Phytoplasma 16S rDNA sequences retrieved from the GenBank and employed in phylogenetic analyses in this study.

IsolatePhytoplasma

species

16S rRNAGroup-

subgroupHost species Location

NCBIAccession No.

Literature

Aster yellows Ca. P asteris 16SrI Cannabis sativa L India EU439257.1 Raj et al, 2008

Napier grass stunt Ca. P oryzae 16SrXI P. purpuruem Kenya, Mbita FJ862999.2 Obura et al, 2009

Napier grass stunt Ca. P oryzae 16SrXI P. purpuruem Kenya, Bungoma FJ862997.2 Obura et al, 2009

Bermuda grass white leaf Ca. P cynodontis 16SrXIV Cynodon dactylon China EU999999.1 Li et al, 2008

Rice yellow Dwarf Ca. P oryzae 16SrXI Oryza sativa Vietnam JF927999.1 Trinh et al, 2011

Peanut witches’ - broom Ca. P aurantifolia 16SrII Citrus araurantifolia Oman, Rumis AB295060.1 Natsuaki & Al-Zadjali, 2007

X-disease Ca. P pruni 16SrIII-A stone fruits, Prunus U.S.A/ canada JQ044393.1 Davies R. E., 2011

Stolbur Ca. P solani 16SrXII-A Solanum tuberosum  Romania/ Russia HQ108391.1 Ember et al, 2011

Elm yellows Ca. P ulmi 16SrV-A Ulmus spp Serbia HM038459.1 Jovic et al, 2011

Clover proliferation Ca. P trifolii 16SrVI Calotropis gigantean India: Gorakhpur HM485690.1 Priya et al, 2010

Ash yellows Ca. P fraxini 16SrVIIA Graminella nigrifrons Canada JN563608.1 Arocha-Rosete, 2011

Pigeonpea witches'-broom Ca. P phoenicium 16SrIX Blueberry U.S.A JN791267.1 Lee et al, 2012

Apple proliferation Ca. P mali Gn-16SrXA Graminella nigrifrons Canada JN563610.1 Arocha-Rosete, 2011

Apple proliferation Ca. P pyri 16SrX Cacopsylla pyri Portugal JN644986.1 Sousa et al, 2011

Mexican periwinkle viresc Unidentified 16SrXIII-A Catharanthus roseus U.S.A AF248960.1 Dally et al, 2000

Bermuda grass white leaf Ca. P cynodontis 16SrXIV Dicanthium annulatum India FJ348654.1 Rao et al, 2008

Hibiscus witches'-broom Ca. P brasiliense 16SrXV Prunus persica Azerbaijan FR717540.1 Balakishiyeva et al, 2010

A dendogram of partial 16S rRNA gene sequences from 33 wild grass phytoplasmas from western Kenya

Grass speciesPCR status Percentage

of infection Total

0 1Brachiaria brizantha 71(0.8452) 13(0.1548) 13(16.0000) 84

Cenchrus ciliaris 0 1(1.0000) 1(1.2000) 1

Cymbopogon nardus 2(1.0000) 0 0 2

Cynodon dactylon 55(0.6395) 31(0.3605) 31(38.3000) 86

Digitaria scalarum 286(0.9533) 14(0.0467) 14(17.3000) 300

Echinichloa pyramidalis 2(1.0000) 0 0 2

Eleusine indica 6(0.7500) 2(0.2500) 2(2.5000) 8

Eragrostis curvula 4(1.0000) 0 0 4

Hyparrhenia pilgerama 6(1.0000) 0 0 6

Other 65(0.8784) 9(0.1216) 9(11.1000) 74

Panicum maximum 28(0.8750) 4(0.1250) 4(4.9000) 32

Pennisetum polystachion 5(1.0000) 0 0 5

Pennisetum purpureum 1(1.0000) 0 0 1

Poverty grass 24(0.8000) 6(0.2000) 6(7.4000) 30

R. cochinchinensis 1(1.0000) 0 0 1

Setaria incrassata 2(0.6667) 1(0.3333) 1(1.2000) 3

Sorghum versicolor 2(1.0000) 0 0 2

Sporobolus pyramidalis 4(1.0000) 0 0 4

Themeda triada 1(1.0000) 0 0 1

Total 565 81 81(100) 646Chi square test 75.787(a)

df 18

Likelihood Ratio 68.054

P Value (≤0.05) 0.001

Total grass species, their phytoplasma statuses and the proportions of infection

grass species collected and their associated 16S rRNA sub-group

Grass species 16SrXI 16SrXIV Not done Total

B. brizantha 4(57.14%) 3(42.86%) 77 84

C. ciliaris 0 0 1 1

C. nardus 0 0 2 2

C. dactylon 2(18.18%) 9(81.81%) 75 86

D. scalarum 6(100%) 0 294 300

E. pyramidalis 0 0 2 2

E. indica 1(100%) 0 7 8

E. curvula 0 0 4 4

H. pilgerama 0 0 6 6

Other 2(100%) 0 72 74

P. maximum 3(100%) 0 29 32

P. polystachion 0 0 5 5

P. purpureum 0 0 1 1

Poverty grass 3(100%) 0 27 30

R. cochinchinensis 0 0 1 1

S. incrassata 0 0 3 3

S. versicolor 0 0 2 2

S. pyramidalis 0 0 4 4

T. triada 0 0 1 1

Total 21 12 613 646

Association between Phytoplasma strains and Grass species

Association between Phytoplasma strains and Locations of survey

Inferences• There is great diversity of wild grasses in Busia and Bungoma districts with D.

scalarum, C. dactylon, B. brizantha grasses being most abundant: (72.5%) • 63% of all sampled phytoplasma positive grasses had latent infections

• C. dactylon, D. scalarum and B. brizantha had the highest proportions of infections at 38%, 17.3% and 16% respectively

• C. dactylon and B. brizantha grass species were the only grasses that

registered phytoplasma sub-group 16SrXIV infections

• 16SrXI group of phytoplasma infects wide range of grasses; D. scalarum, P. maximum, E. indica, poverty grass and to a lesser extent C. dactylon and B. brizantha.

Conclusions1. Phytoplasma is widespread in many local wild grass populations in Busia

and Bungoma counties

2. Approximately more than half of phytoplasma infections are latent/ assymptomatic

3. C. dactylon, B. brizantha, D. scalarum, P. maximum and poverty grass act as wild phytoplasma hosts and are abundantly distributed

4. Phytoplasma sub-groups 16SrXI and 16SrXIV are the only groups infecting wild grasses in western Kenya. (No novel strain)

Recommendations

• Establish a buffer zone clear of wild grasses around Napier fields to reduce the risk of phytoplasma inoculation

• Conduct back-transmission studies to confirm transmissibility of Ns phytoplasma to Napier grass wild grasses

• Carry out a wider survey with a larger sample size to fully understand the dynamics of Ns disease

• Need to genetically engineer napier grass for the production of transgenic tolerant clones against Ns disease

Acknowledgement

• KENYATTA UNIVERSITY• SUPERVISORS• I.C.I.PE

Thank you